TRIM5? is a cytoplasmic restriction factor that inhibits infection by retroviruses by other species. Specifically, TRIM5? proteins from rhesus monkeys can inhibit infection by HIV-1, although the mechanism by which this inhibition occurs remains poorly understood. This application seeks to examine the interaction between retroviruses and cytoplasmic restriction factors microscopically. The research will expand upon previously developed fluorescent microscopy methods that allow the identification of HIV-1 virions that have productively entered the target cell cytoplasm. Using this system, we have shown an interaction between cytoplasmic HIV-1 virions and TRIM5? protein from rhesus macaque during restriction. This work will examine the interaction between retroviruses, including HIV-1 and MLV, and restriction factors, including TRIM5? and Fv-1, to more precisely understand the events leading to retroviral restriction. This will be accomplished by examining the ability of relevant TRIM5? mutants to interact with cytoplasmic virions and restrict infection. As the interaction between restriction factors and retroviruses occurs between individual mature capsid cores and cytoplasmic restriction factors, this interaction has proven very difficult to examine biochemically. Direct observation of this interaction microscopically therefore represents an extremely promising method by which to examine this process. A better understanding of how restriction factors inhibit infection by retroviruses could lead to the development of pharmaceuticals designed to intervene at this currently unexploited step in the viral life cycle. Lay language description: Recently identified primate proteins called restriction factors have recently been found to inhibit infection by viruses native to other species. The TRIM5? protein from rhesus monkeys has recently been identified as being able to inhibit infection by HIV-1. This research application seeks to better understand the mechanism by which this restriction occurs with the hope that this understanding leads to better treatment of people infected with HIV-1 or methods to prevent the spread of the virus.